The overall object of this project is to understand the mechanism of genome expression in eukaryotic organisms. Whereas much emphasis has been placed on the role of chromatin proteins in controlling gene expression, very little is known about the role of DNA modification in this process. We intend to approach this question from both directions by examining the conformation of the active regions of chromatin and by studying eukaryotic DNA methylation in order to illucidate its biological role. A. Using a sophisticated method to specifically label the active regions of the nucleus we intend to analyze those factors which characterize the special conformation of the transcriptionally active genes. A modification of this technique will permit us to label metaphase chromosomes in situ and thus enable us to visualize these regions with regard to chromosome morphology. B. With the aid of some new restriction enzyme technology we can now study both the location and quantity of 5mCyt in eukaryotic DNA. This methodology will also enable us to study the process of methylation. By inserting specific methylated and non methylated genes into eukaryotic cells in culture through the process of transfection, we will be able to follow the metabolic fate of this modification. This technique provides a sort of "in vivo test tube" for studying the biological role of DNA methylation.